The blood-brain barrier (BBB) represents a physical, transport, and metabolic barrier between the bloodstream and the brain and its function is crucial to maintain brain homeostasis. BBB dysfunction is a hallmark of many neurological diseases and disorders. Moreover, the BBB prevents treatment of central nervous system diseases by limiting brain uptake of small molecule and protein-based pharmaceuticals. In vitro models of the BBB provide tools to understand BBB structure and function during development and disease and facilitate discovery of strategies to delivery pharmaceuticals to the brain. Established in vitro BBB models often lack key physiologic phenotypes of the in vivo BBB, however, limiting their utility. Previously, we described a defined method for directed differentiation of human induced pluripotent stem cells (iPSCs) to brain microvascular endothelial cells (BMECs) that comprise the BBB. These iPSC-BMECs express BBB-specific markers and exhibit barrier and transporter properties similar to those in the BBB in vivo, albeit at reduced levels. Our preliminary data demonstrate that application of shear flow to iPSC-BMEC progenitors induces BBB phenotypes in a p21 and TGF? signaling pathway dependent manner. These data motivate our central hypothesis: Shear stress enhances development and maintenance of BBB barrier and transporter phenotypes in iPSC-BMEC progenitors via p21 and TGF? signaling. To test this hypothesis, we will apply shear flow to iPSCs differentiating to BMECs at different stages of development and quantify effects of shear stress on BBB barrier and transporter phenotypes in the resulting BMECs. We will employ genetic and biochemical inhibitors to elucidate the roles of p21 and TGF? pathway induction of BBB phenotypes. Based on these fundamental studies, we will construct isogenic, neurovascular unit (NVU) models comprised of shear-conditioned iPSC- derived BMECs, neurons, astrocytes and pericytes that will enable a better understanding of human BBB development and disease and facilitate neurotherapeutic development. Our specific aims are: 1. Quantify the effects of shear stress applied to iPSC-BMEC progenitors on induction of BBB phenotypes. We will assess the developmental stages at which shear induces barrier and transporter phenotypes in differentiating iPSC-BMECs. 2. Elucidate the roles of p21 and TGF? signaling in shear-mediated induction of BBB phenotypes in iPSC- BMEC progenitors. We will employ chemical and genetic inhibition of p21 and TGF? pathways to test the necessity of these pathways in shear induction of BBB phenotypes in iPSC-BMEC progenitors. 3. Evaluate shear-conditioned iPSC-BMECs in contact and noncontact isogenic neurovascular unit models. We will construct NVU models consisting of iPSC-BMECs differentiated in the presence of shear, and iPSC-derived neurons, astrocytes, and pericytes, to test the hypothesis that shear application to iPSC- BMEC progenitors will yield a high-fidelty NVU model with enhanced, sustained BBB phenotypes.